Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

in Human Genomics (2023), 17(1), 39

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main ... [more ▼]

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques. [less ▲]

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

in Genetics in Medicine (2023), 25(4), 100018

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a ... [more ▼]

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock. [less ▲]

DYT6 mutated THAP1 is a cell type dependent regulator of the SP1 family.

in Brain : a journal of neurology (2022), 145(11), 3968-3984

DYT6 dystonia is caused by mutations in the transcription factor THAP1. THAP1 knock-out or knock-in mouse models revealed complex gene expression changes, which are potentially responsible for the ... [more ▼]

DYT6 dystonia is caused by mutations in the transcription factor THAP1. THAP1 knock-out or knock-in mouse models revealed complex gene expression changes, which are potentially responsible for the pathogenesis of DYT6 dystonia. However, how THAP1 mutations lead to these gene expression alterations and whether the gene expression changes are also reflected in the brain of THAP1 patients are still unclear. In this study we used epigenetic and transcriptomic approaches combined with multiple model systems [THAP1 patients' frontal cortex, THAP1 patients' induced pluripotent stem cell (iPSC)-derived midbrain dopaminergic neurons, THAP1 heterozygous knock-out rat model, and THAP1 heterozygous knock-out SH-SY5Y cell lines] to uncover a novel function of THAP1 and the potential pathogenesis of DYT6 dystonia. We observed that THAP1 targeted only a minority of differentially expressed genes caused by its mutation. THAP1 mutations lead to dysregulation of genes mainly through regulation of SP1 family members, SP1 and SP4, in a cell type dependent manner. Comparing global differentially expressed genes detected in THAP1 patients' iPSC-derived midbrain dopaminergic neurons and THAP1 heterozygous knock-out rat striatum, we observed many common dysregulated genes and 61 of them were involved in dystonic syndrome-related pathways, like synaptic transmission, nervous system development, and locomotor behaviour. Further behavioural and electrophysiological studies confirmed the involvement of these pathways in THAP1 knock-out rats. Taken together, our study characterized the function of THAP1 and contributes to the understanding of the pathogenesis of primary dystonia in humans and rats. As SP1 family members were dysregulated in some neurodegenerative diseases, our data may link THAP1 dystonia to multiple neurological diseases and may thus provide common treatment targets. [less ▲]

Unraveling Molecular Mechanisms of THAP1 Missense Mutations in DYT6 Dystonia

in Journal of Molecular Neuroscience (2020)

Mutations in THAP1 (THAP domain-containing apoptosis-associated protein 1) are responsible for DYT6 dystonia. Until now, more than eighty differentmutations in THAP1 gene have been found in patientswith ... [more ▼]

Mutations in THAP1 (THAP domain-containing apoptosis-associated protein 1) are responsible for DYT6 dystonia. Until now, more than eighty differentmutations in THAP1 gene have been found in patientswith primary dystonia, and two third of them are missense mutations. The potential pathogeneses of these missense mutations in human are largely elusive. In the present study, we generated stable transfected human neuronal cell lines expressing wild-type or mutated THAP1 proteins found in DYT6 patients. Transcriptional profiling using microarrays revealed a set of 28 common genes dysregulated in two mutated THAP1 (S21T and F81L) overexpression cell lines suggesting a common mechanism of these mutations. ChIP-seq showed that THAP1 can bind to the promoter of one of these genes, superoxide dismutase 2 (SOD2). Overexpression of THAP1 in SK-N-AS cells resulted in increased SOD2 protein expression, whereas fibroblasts from THAP1 patients have less SOD2 expression, which indicates that SOD2 is a direct target gene of THAP1. In addition, we show that some THAP1 mutations (C54Y and F81L) decrease the protein stability which might also be responsible for altered transcription regulation due to dosage insufficiency. Taking together, the current study showed different potential pathogenic mechanisms of THAP1 mutations which lead to the same consequence of DYT6 dystonia. [less ▲]

Metformin reverses TRAP1 mutation-associated alterations in mitochondrial function in Parkinson's disease

in Brain : A Journal of Neurology (2017), 140(9), 2444-2459

The mitochondrial proteins TRAP1 and HtrA2 have previously been shown to be phosphorylated in the presence of the Parkinson’s disease kinase PINK1 but the downstream signaling is unclear. HtrA2 and PINK1 ... [more ▼]

The mitochondrial proteins TRAP1 and HtrA2 have previously been shown to be phosphorylated in the presence of the Parkinson’s disease kinase PINK1 but the downstream signaling is unclear. HtrA2 and PINK1 loss of function causes parkinsonism in humans and animals. Here, we identified TRAP1 as an interactor of HtrA2 using an unbiased mass spectrometry approach. In our human cell models, TRAP1 overexpression is protective, rescuing HtrA2 and PINK1-associated mitochondrial dysfunction and suggesting that TRAP1 acts downstream of HtrA2 and PINK1. HtrA2 regulates TRAP1 protein levels, but TRAP1 is not a direct target of HtrA2 protease activity. Following genetic screening of Parkinson’s disease patients and healthy controls, we also report the first TRAP1 mutation leading to complete loss of functional protein in a patient with late onset Parkinson’s disease. Analysis of fibroblasts derived from the patient reveal that oxygen consumption, ATP output and reactive oxygen species are increased compared to healthy individuals. This is coupled with an increased pool of free NADH, increased mitochondrial biogenesis, triggering of the mitochondrial unfolded protein response, loss of mitochondrial membrane potential and sensitivity to mitochondrial removal and apoptosis. These data highlight the role of TRAP1 in the regulation of energy metabolism and mitochondrial quality control. Interestingly, the diabetes drug metformin reverses mutation-associated alterations on energy metabolism, mitochondrial biogenesis and restores mitochondrial membrane potential. In summary, our data show that TRAP1 acts downstream of PINK1 and HtrA2 for mitochondrial fine tuning, whereas TRAP1 loss of function leads to reduced control of energy metabolism, ultimately impacting mitochondrial membrane potential. These findings offer new insight into mitochondrial pathologies in Parkinson’s disease and provide new prospects for targeted therapies. [less ▲]

Overexpression of synphilin-1 promotes clearance of soluble and misfolded alpha-synuclein without restoring the motor phenotype in aged A30P transgenic mice.

in Human molecular genetics (2014), 23(3), 767-81

Lewy bodies and neurites are the pathological hallmark of Parkinson's disease. These structures are composed of fibrillized and ubiquitinated alpha-synuclein suggesting that impaired protein clearance is ... [more ▼]

Lewy bodies and neurites are the pathological hallmark of Parkinson's disease. These structures are composed of fibrillized and ubiquitinated alpha-synuclein suggesting that impaired protein clearance is an important event in aggregate formation. The A30P mutation is known for its fast oligomerization, but slow fibrillization rate. Despite its toxicity to neurons, mechanisms involved in either clearance or conversion of A30P alpha-synuclein from its soluble state into insoluble fibrils and their effects in vivo are poorly understood. Synphilin-1 is present in Lewy bodies, interacting with alpha-synuclein in vivo and in vitro and promotes its sequestration into aggresomes, which are thought to act as cytoprotective agents facilitating protein degradation. We therefore crossed animals overexpressing A30P alpha-synuclein with synphilin-1 transgenic mice to analyze its impact on aggregation, protein clearance and phenotype progression. We observed that co-expression of synphilin-1 mildly delayed the motor phenotype caused by A30P alpha-synuclein. Additionally, the presence of N- and C-terminal truncated alpha-synuclein species and fibrils were strongly reduced in double-transgenic mice when compared with single-transgenic A30P mice. Insolubility of mutant A30P and formation of aggresomes was still detectable in aged double-transgenic mice, paralleled by an increase of ubiquitinated proteins and high autophagic activity. Hence, this study supports the notion that co-expression of synphilin-1 promotes formation of autophagic-susceptible aggresomes and consecutively the degradation of human A30P alpha-synuclein. Notably, although synphilin-1 overexpression significantly reduced formation of fibrils and astrogliosis in aged animals, a similar phenotype is present in single- and double-transgenic mice suggesting additional neurotoxic processes in disease progression. [less ▲]

Role of sepiapterin reductase gene at the PARK3 locus in Parkinson's disease.

in Neurobiology of Aging (2011), 32(11), 21081-5

Sepiapterin reductase (SPR) gene is an enzyme which catalyses the final step of tetrahydrobiopterin synthesis (BH4) and was implicated in Parkinson's disease (PD) pathogenesis as a candidate gene for ... [more ▼]

Sepiapterin reductase (SPR) gene is an enzyme which catalyses the final step of tetrahydrobiopterin synthesis (BH4) and was implicated in Parkinson's disease (PD) pathogenesis as a candidate gene for PARK3 locus. A number of studies yielded association of the PARK3 locus with PD, and SPR knockout mice were shown to display parkinsonian features. To evaluate the role of SPR gene polymorphisms in diverse populations in PD, we performed collaborative analyses in the Genetic Epidemiology of Parkinson Disease (GEO-PD) Consortium. A total of 5 single nucleotide polymorphisms (3 in the promoter region and 2 in the 3' untranslated region [UTR]) were genotyped. Fixed as well as random effect models were used to provide summary risk estimates of SPR variants. A total of 19 sites provided data for 6547 cases and 9321 controls. Overall odds ratio estimates varied from 0.92 to 1.01. No overall association with the SPR gene using either fixed effect or random effect model was observed in the studied population. I(2) Metric varied from 0% to 36.2%. There was some evidence for an association for participants of North European/Scandinavian descent with the strongest signal for rs1876487 (odds ratio = 0.82; p value = 0.003). Interestingly, families which were used to map the PARK3 locus, have Scandinavian ancestry suggesting a founder effect. In conclusion, this large association study for the SPR gene revealed no association for PD worldwide. However, taking the initial mapping of the PARK3 into account, the role of a population-specific effect warrants consideration in future studies. [less ▲]

Olfactory neuron-specific expression of A30P alpha-synuclein exacerbates dopamine deficiency and hyperactivity in a novel conditional model of early Parkinson's disease stages.

in Neurobiology of Disease (2011), 44(2), 192-204

Mutations in the N-terminus of the gene encoding alpha-synuclein (alpha-syn) are linked to autosomal dominantly inherited Parkinson's disease (PD). The vast majority of PD patients develop ... [more ▼]

Mutations in the N-terminus of the gene encoding alpha-synuclein (alpha-syn) are linked to autosomal dominantly inherited Parkinson's disease (PD). The vast majority of PD patients develop neuropsychiatric symptoms preceding motor impairments. During this premotor stage, synucleinopathy is first detectable in the olfactory bulb (OB) and brain stem nuclei; however its impact on interconnected brain regions and related symptoms is still less far understood. Using a novel conditional transgenic mouse model, displaying region-specific expression of human mutant alpha-syn, we evaluated effect and reversibility of olfactory synucleinopathy. Our data showed that induction of mutant A30P alpha-syn expression increased transgenic deposition into somatodendritic compartment of dopaminergic neurons, without generating fibrillar inclusions. We found reversibly reduced levels of dopamine and metabolites in the OB, suggesting an impact of A30P alpha-syn on olfactory neurotransmitter content. We further showed that mutant A30P expression led to neurodegenerative changes on an ultrastructural level and a behaviorally hyperactive response correlated with novelty, odor processing and stress associated with an increased dopaminergic tone in midbrain regions. Our present data indicate that mutant (A30P) alpha-syn is directly implicated in reduction of dopamine signaling in OB interneurons, which mediates further alterations in brain regions without transgenic expression leading functionally to a hyperactive response. These modulations of neurotransmission may underlie in part some of the early neuropsychiatric symptoms in PD preceding dysfunction of the nigrostriatal dopaminergic system. [less ▲]

Reduced basal autophagy and impaired mitochondrial dynamics due to loss of Parkinson's disease-associated protein DJ-1.

in PloS one (2010), 5(2), 9367

BACKGROUND: Mitochondrial dysfunction and degradation takes a central role in current paradigms of neurodegeneration in Parkinson's disease (PD). Loss of DJ-1 function is a rare cause of familial PD ... [more ▼]

BACKGROUND: Mitochondrial dysfunction and degradation takes a central role in current paradigms of neurodegeneration in Parkinson's disease (PD). Loss of DJ-1 function is a rare cause of familial PD. Although a critical role of DJ-1 in oxidative stress response and mitochondrial function has been recognized, the effects on mitochondrial dynamics and downstream consequences remain to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Using DJ-1 loss of function cellular models from knockout (KO) mice and human carriers of the E64D mutation in the DJ-1 gene we define a novel role of DJ-1 in the integrity of both cellular organelles, mitochondria and lysosomes. We show that loss of DJ-1 caused impaired mitochondrial respiration, increased intramitochondrial reactive oxygen species, reduced mitochondrial membrane potential and characteristic alterations of mitochondrial shape as shown by quantitative morphology. Importantly, ultrastructural imaging and subsequent detailed lysosomal activity analyses revealed reduced basal autophagic degradation and the accumulation of defective mitochondria in DJ-1 KO cells, that was linked with decreased levels of phospho-activated ERK2. CONCLUSIONS/SIGNIFICANCE: We show that loss of DJ-1 leads to impaired autophagy and accumulation of dysfunctional mitochondria that under physiological conditions would be compensated via lysosomal clearance. Our study provides evidence for a critical role of DJ-1 in mitochondrial homeostasis by connecting basal autophagy and mitochondrial integrity in Parkinson's disease. [less ▲]

Dissecting the role of the mitochondrial chaperone mortalin in Parkinson's disease: functional impact of disease-related variants on mitochondrial homeostasis.

in Human molecular genetics (2010), 19(22), 4437-52

The mitochondrial chaperone mortalin has been linked to neurodegeneration in Parkinson's disease (PD) based on reduced protein levels in affected brain regions of PD patients and its interaction with the ... [more ▼]

The mitochondrial chaperone mortalin has been linked to neurodegeneration in Parkinson's disease (PD) based on reduced protein levels in affected brain regions of PD patients and its interaction with the PD-associated protein DJ-1. Recently, two amino acid exchanges in the ATPase domain (R126W) and the substrate-binding domain (P509S) of mortalin were identified in Spanish PD patients. Here, we identified a separate and novel variant (A476T) in the substrate-binding domain of mortalin in German PD patients. To define a potential role as a susceptibility factor in PD, we characterized the functions of all three variants in different cellular models. In vitro import assays revealed normal targeting of all mortalin variants. In neuronal and non-neuronal human cell lines, the disease-associated variants caused a mitochondrial phenotype of increased reactive oxygen species and reduced mitochondrial membrane potential, which were exacerbated upon proteolytic stress. These functional impairments correspond with characteristic alterations of the mitochondrial network in cells overexpressing mutant mortalin compared with wild-type (wt), which were confirmed in fibroblasts from a carrier of the A476T variant. In line with a loss of function hypothesis, knockdown of mortalin in human cells caused impaired mitochondrial function that was rescued by wt mortalin, but not by the variants. Our genetic and functional studies of novel disease-associated variants in the mortalin gene define a loss of mortalin function, which causes impaired mitochondrial function and dynamics. Our results support the role of this mitochondrial chaperone in neurodegeneration and underscore the concept of impaired mitochondrial protein quality control in PD. [less ▲]